Abstract

Background

Recent incidents, such as the SARS and influenza epidemics, have highlighted the need
for readily available antiviral drugs. One important precursor currently used for
the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic
acid (NeuNAc). This substance has a considerably high market price despite efforts
to develop cost-reducing (biotechnological) production processes. Hypocrea jecorina (Trichoderma reesei) is a saprophyte noted for its abundant secretion of hydrolytic enzymes and its potential
to degrade chitin to its monomer N-acetylglucosamine (GlcNAc). Chitin is considered
the second most abundant biomass available on earth and therefore an attractive raw
material.

Results

In this study, we introduced two enzymes from bacterial origin into Hypocrea, which convert GlcNAc into NeuNAc via N-acetylmannosamine. This enabled the fungus
to produce NeuNAc from the cheap starting material chitin in liquid culture. Furthermore,
we expressed the two recombinant enzymes as GST-fusion proteins and developed an enzyme
assay for monitoring their enzymatic functionality. Finally, we demonstrated that
Hypocrea does not metabolize NeuNAc and that no NeuNAc-uptake by the fungus occurs, which are
important prerequisites for a potential production strategy.

Conclusions

This study is a proof of concept for the possibility to engineer in a filamentous
fungus a bacterial enzyme cascade, which is fully functional. Furthermore, it provides
the basis for the development of a process for NeuNAc production as well as a general
prospective design for production processes that use saprophytes as whole-cell catalysts.